(1) Field of the Invention
This invention relates generally to the field of breast cancer pathogenesis and, more particularly, to a cDNA sequence and encoded mammary-specific protein for use in detecting and treating breast cancer.
(2) Description of the Related Art
Breast cancer is one of the most common and potentially lethal of cancers. Although early diagnosis and treatment can reduce morbidity and mortality related to the disease, the positive predictive value of mammography has been estimated to be only about 25% (Hall et al., N Engl J Med 327:319-328, 1992). It would, therefore, be desirable to have a means for detecting the cancer earlier than the cancer can be detected using mammography and a genetic or biochemical marker might be able to provide such means to complement and increase the predictive value of mammography. (Hayes, Hematol Oncol Clin N Am 8:485, 1994).
The development of breast cancer is accompanied by a number of genetic changes (For review see Porter-Jordan, Hematol Oncol Clin N Am 8:73, 1994). Such changes include gross chromosomal alterations and loss of genetic markers (Devilee et al, Biochim Biophys Acta 1198:113, 1994; Callahan et al, J Cell Biochem Suppl 17:167, 1993). The progression of breast neoplasia has also been shown to result in qualitative and quantitative changes in expression of previously identified genes that encode growth factors and their receptors (Zajchowski et al., Cancer Res 48:7041, 1988), structural proteins (Trask et al., Proc Natl Acad Sci 87:2319, 1990), second messenger proteins (Ohuchi et al., Cancer Res 26:2511, 1986), and transcription factors (Harris, Adv Cancer Res 59:69:1992). These changes in gene expression could potentially form the basis for developing a breast cancer marker, although the precise role of these gene changes in the pathogenesis of breast carcinoma in patient biopsy samples is not well understood.
In addition to providing a genetic or biochemical marker for breast cancer for early detection of the disease, it would also be desirable to have a tumor marker that might provide an estimation of prognosis, a means for selection and evaluation of therapy and a means for the targeting of therapy. Although a number of tissue markers have been identified, none are sufficiently sensitive or tumor specific to be ideally suited for diagnosis or for screening the general population. (Id.) Thus, there remains a continuing need for a breast cancer marker such as a gene along with its expressed protein that can be used to specifically and selectively identify the appearance and pathogenic development of breast cancer in a patient, and that can be used in tumor-specific immunotherapy.
Using a modified differential display polymerase chain reaction technique to isolate differentially expressed sequence tags from mammary carcinoma, several sequence fragments were isolated that were uniquely expressed in neoplastic mammary epithelial tissue as compared to normal tissue controls (Watson and Fleming, Cancer Res 54:4598-4602, 1994). The discovery of one of these sequence tags identified as DEST002 has led to the discovery and isolation of the novel full length cDNA and encoded protein now referenced as mammaglobin. The cDNA and protein are both new.
Briefly, therefore, the present invention is directed to the identification of novel genes whose expression is increased in breast cancer and to the isolating of cDNA""s from the mRNA""s of these genes. Accordingly, applicants have succeeded in discovering a novel cDNA and the encoded mammary-specific secretory protein, mammaglobin. The cDNA is in purified and isolated form and comprises a nucleotide sequence identified as SEQ ID NO:15 and the encoded protein, mammaglobin, is in purified and isolated form and has an amino acid sequence identified as SEQ ID NO:2.
In a small-scale study described in U.S. Pat. No. 5,668,267, mammaglobin mRNA was overexpressed in 27% of stage I primary breast cancer tumors. The present application describes a larger survey of primary breast tumors of multiple grades and histological types in which mammaglobin protein was detected in about 80% of the tumors examined. These data suggest that dysregulation of the mammaglobin gene occurs early and frequently in breast cancer. The discovery of mammaglobin and its cDNA, therefore, provide the basis for the development of novel methods and compositions for the detection and treatment of breast neoplastic disease in humans and other mammals.
Thus, the present invention is directed to novel methods for detecting the presence of breast neoplasia cells in a sample. In one embodiment, a polynucleotide probe is used to detect the presence of mammaglobin mRNA in the sample. The method comprises the steps of: (a) contacting mRNA in the sample with a polynucleotide probe which specifically hybridizes to a mammaglobin mRNA comprising SEQ ID NO:15 or an allelic variant thereof, and (b) detecting a hybridization complex between the probe and the sample mRNA.
Another aspect of the present invention provides a kit for detecting the presence of breast neoplasia cells in a sample by hybridization. The kit comprises a polynucleotide probe which specifically hybridizes to a mammaglobin mRNA comprising SEQ ID NO:15 or an allelic variant thereof packaged in a container.
In another embodiment of the present invention, mammaglobin expression in a sample is determined by detecting the presence of cDNA that is reverse transcribed from mammaglobin mRNA in the sample. The method comprises the steps of: (a) producing a cDNA encoding mammaglobin from mRNA using the reverse transcription method in a sample obtained from a patient, (b) providing two primers for the polymerase chain reaction method which comprise oligomers that flank or lie within the cDNA encoding mammaglobin, and (c) amplifying the cDNA encoding mammaglobin by the polymerase chain reaction method. Preferably, the two primers have nucleotide sequences encoding SEQ ID NO:4 and SEQ ID NO:16.
Another embodiment to the present invention provides a kit for detecting the presence of breast neoplasia cells in a sample by the polymerase chain reaction. The kit comprises two primers for the polymerase chain reaction method which comprise oligomers that flank or lie within a cDNA encoding mammaglobin packaged in a container. Preferably, the two primers have nucleotide sequences comprising SEQ ID NO:4 and SEQ ID NO:16.
In another embodiment of the present invention, the presence of mammaglobin protein expressed by a tumor cell is detected in a sample using specific antibodies to the mammaglobin protein. The specific antibodies can be polyclonal or monoclonal antibodies.
The invention is also directed to novel compositions and methods for treating breast neoplastic disease using mammaglobin antigens capable of inducing an antibody-mediated and/or a cell-mediated, i.e., through activated T cells, immune response against a mammaglobin-expressing tumor.
One embodiment of a composition according to the invention comprises a mammaglobin B cell antigen capable of activating mammaglobin-specific B cells. The B cell antigen comprises a mammaglobin-specific B cell epitope and a TH epitope, or determinant, recognized by T helper cells.
In another embodiment, the mammaglobin antigen is a mammaglobin TC cell antigen recognized by mammaglobin-specific cytotoxic T lymphocytes which comprises a TC cell epitope and a binding site, or agretope, for a MHC class I molecule.
Yet another embodiment of a composition according to the invention comprises B cell and TC cell antigens.
Methods for treating a patient with a mammaglobin-expressing tumor include adoptive immunotherapy, which comprises ex vivo stimulation with a mammaglobin antigen of mammaglobin-specific lymphocytes isolated from the patient and subsequent administration of the activated lymphocytes to the patient, and in vivo stimulation of an anti-mammaglobin immune response, which comprises administering to the patient a vaccine comprising a mammaglobin antigen.
Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of a nucleotide sequence and encoded amino acid sequence that can serve as markers for breast cancer cells; the provision of methods for early detection of the presence of breast neoplasia cells; the provision of means for detecting breast cancer that can complement mammography and increase the predictive value; the provision of methods that can provide an estimation of prognosis; the provision of markers that will allow the targeting of therapy; and the provision of compositions for stimulating a cellular and humoral immune response against the tumor.